JP2001522799A - Method for producing arsenic trioxide preparation and method for treating cancer using arsenic trioxide or melarsoprol - Google Patents

Abstract

  (57) [Summary]     The present invention relates to the use of arsenic compounds for treating various leukemias, lymphomas and solid tumors. Furthermore, the arsenic compounds can be used in combination with other therapeutic agents such as retinoids. The present invention also provides a method for producing an arsenic trioxide preparation.

Description

DETAILED DESCRIPTION OF THE INVENTION

1. FIELD OF THE INVENTION The present invention relates to methods and compositions for the treatment of leukemias, lymphomas and certain other cancers.

More specifically, the present invention relates to novel uses of arsenic trioxide and organic arsenic compounds for treating acute and chronic leukemias.

[0003] 2. BACKGROUND OF THE INVENTION 2.1. Cancer Cancer is primarily increased number of abnormal cells derived from a certain normal tissues, invasion of adjacent tissues by these abnormal cells, and regional lymph nodes and lymphatic or blood-borne spread of malignant cells to distant sites (Metastasis). Clinical data and molecular biology studies indicate that cancer is a multistep process that begins with small pre-tumor changes and progresses under certain conditions to a neoplasm.

Prior to the formation of neoplastic lesions, precancerous abnormal cell growth occurs, exemplified by hyperplasia, dysplasia and dysplasia (for a review of such abnormal growth states, see Robbins and Angell, 1976, Basic Pathology, 2nd edition, WB Saunders Co., Philadelphi
a, pp. 68-79). Neoplastic lesions can clonally form and grow into solid tumors, progressively developing their capacity for invasion, growth, metastasis and heterogeneity (particularly in conditions where neoplastic cells escape host immune surveillance). (Roitt, I.,
Brostoff, J and Kale, D., 1993, Immunology, Third Edition, Mosby, St. Louis.p.
ps.17.1-17.12).

[0005] Leukemia refers to a malignant neoplasm of blood forming tissue. Transformation to a malignant state typically occurs in more than one stage in a single cell, followed by proliferation and clonal expansion (clonal expansion). In some leukemias, specific chromosomal translocations have been identified with consistent leukemia cell morphology and special clinical features (eg, translocations 9 and 22 in chronic myeloid leukemia, Translocations 15 and 17 in acute promyelocytic leukemia).
In acute leukemia, the undifferentiated cell population predominates, and in chronic leukemia, the more mature cell morphology predominates.

[0006] Acute leukemias are classified into lymphoblastoid (ALL) and non-lymphoblastic (ANLL) types. They can be further subdivided according to their morphological and cytochemical appearance according to the FAB (French-American-British) classification or according to their type and degree of differentiation. For classification, the use of specific B and T cells and myeloid-antigen monoclonal antibodies is most useful. ALL is a childhood disease primarily confirmed by laboratory findings and bone marrow examination. ANLL, also known as acute myeloblastic leukemia (AML), occurs at any age and is a more common acute leukemia in adults. It is a form that usually involves irradiation as a causative factor.

[0007] Chronic leukemia is described as lymphocytic (CLL) or myeloid (CML). CL
L is characterized by the appearance of mature lymphocytes in blood, bone marrow and lymphoid organs. CLL is characterized by persistent absolute lymphocytosis (> 5,000 / μL) and increased lymphocytes in the bone marrow. Also, most CLL patients have a clonal expansion of lymphocytes with B cell characteristics. CLL is a disease of the elderly. A characteristic feature in CML is the predominance of granulocytes of all stages of differentiation in blood, bone marrow, liver, spleen and other organs. In symptomatic patients at the time of diagnosis, the total WBC count is usually about 200,000 / μL, but can reach 1,000,000 / μL. Diagnosis of CML is relatively easy due to the presence of the Philadelphia chromosome.

[0008] Because of the very nature of hematopoietic cancer, systemic chemotherapy has to be used as the primary treatment modality. Drugs selected for a particular leukemia susceptibility are usually administered in combination. Radiation therapy can additionally be used to address the local accumulation of leukemia cells. Surgery is rarely applied as a primary treatment modality,
It may be used in the treatment of some complications. Bone marrow transplants from HLA-matched siblings may be applied.

[0009] 2.2. Arsenic and its medical uses Arsenic has long been regarded as a toxic and a drug in both Western and Chinese medical practice. In the late 19th century, arsenic was used extensively in the West in attempts to treat blood disorders. It was reported in 1878 that treating leukemia patients with Fowler's solution (a solution containing + 5-valent potassium arsenite) significantly reduced white blood cell counts (Cutler and Bradford, Am. J. Med. Sci. . , 1878
January, 81-84). A further relationship to the use of Fowler's solution as a palliative to treat chronic myeloid leukemia (CML) was described by Forkner and Scott in 1931 ( J. Am. Med. Assoc. , 1931, iii, 97), later confirmed by Stephens and Lawrence in 1936 ( Ann. Intern Med . 9, 1488-1502). However, the effective chemical components of Fowler's solution were not determined, while its toxicity was marked. Fowler's solution is administered exclusively as an oral composition and is administered to leukemia patients as a solution until the level of leukocytes decreases to an acceptable level or until toxicity (eg, cutaneous keratosis and hyperpigmentation) appears, Patients had varying periods of remission. Although Fowler's solution was sometimes used in attempts to treat CML in the 1960s, most CML patients were treated with other chemotherapeutic agents such as busulfan and / or radiation therapy (
Monfardini et al., Cancer , 1973, 31: 492-501).

[0010] Paradoxically, one of the long-recognized effects of exposure to arsenic is skin cancer, which may be of environmental or medical origin. It has nothing to do with it (Hutchinson, 1888, Trans. Path. Soc. Lond. , 39:
352; Neubauer, 1947, Br. J. Cancer , 1: 192). There were even epidemiological data suggesting that long-term use of Fowler's solution may lead to an increased frequency of internal cancer (Cuzick et al . , Br. J. Cancer , 1982, 45: 904-911; Kaspar et al., J. Am. Med. Assoc . , 1984, 252: 3407-3408). Subsequently, due to the fact that arsenic can induce chromosomal abnormalities, gene amplification, sister chromatid exchange and cell transformation,
The carcinogenicity of arsenic has been demonstrated (see, for example, Lee et al., 1988, Science , 241: 79-81; and Germolec et al . , Toxicol. Applied Pharmacol. , 1996, 141: 308-318). Due to the known carcinogenic effects of arsenic, its only therapeutic use in humans in western medicine today is in the treatment of tropical diseases such as African trypanosomiasis (the organic arsenical melarsoprol; Goodman &Gliman's The Pharmacological
Basis of Therapeutics, 9th Edition, Chapter 66, 1659-1662, 1997).

In traditional Chinese medicine, arsenite or arsenic trioxide paste has been used to treat tooth marrow disease, psoriasis, syphilis and rheumatism (Chen et al., 1995, Manual). of Clinical Drugs, Shanghai, China, Shaghai Ins
titute of Science and Technology, p. 830). Arsenic trioxide was applied experimentally to treat acute promyelocytic leukemia (APL) in China in the 1970s (commentary by Mervis, 1996, Science , 273: 578). Recently, the clinical efficacy of arsenic trioxide has been reviewed in 14 out of 15 patients with refractory APL, where intravenous doses of 10 mg / day for 4 to 9 weeks are associated with bone marrow suppression Without complete morphological remission (Shen et al., 1997, Blood , 89: 3354-3360). In addition, arsenic trioxide induces apoptosis (programmed cell death) in vitro in the APL cell line NB4 cells, which downregulates the oncogene bcl-2 and chimeric PML / It has been shown to be associated with intracellular redistribution of RARα protein (Chen et al., 1996, Blood , 88: 1052-1061; Andre et al.
, 1996, Exp. Cell Res. 229: 253-260). The biological activity of arsenic has been reported to be due to the ability of arsenic to lead and degrade the nuclear fraction of PML to nuclear bodies (Zhu et al., 1997, Proc. Natl. Acad. Sci. ., 94: 3978-3983).

Although it is well known that arsenic is both a toxic and a carcinogen, there are numerous reports on the use of arsenic in medical treatment. Furthermore, it should be clear from the foregoing discussion that there are a number of different types of leukemia, each of which requires a unique treatment protocol modified in response to the presence of factors that predict the risk of treatment failure. It is. Thus, there is a great need for the development of broad spectrum anti-leukemia agents that can be used alone or in combination with other existing drugs.

[0013] 3. SUMMARY OF THE INVENTION Despite conflicting reports in the art regarding the benefits and risks of arsenic administration to patients, Applicants believe that arsenic trioxide and the organic arsenic agent melarsoprol could be used to treat various types of leukemia, lymphoma and It has been found to have broad applicability in the treatment of solid tumors.

[0014] The invention described herein is a method of treating leukemia, lymphoma or solid tumor, comprising treating a human in need of such treatment with a non-lethal amount of arsenic trioxide or melarsoprole that is therapeutically effective. A method comprising administering to the subject. The above invention also includes the use of combination therapy to treat leukemia, particularly leukemia that is refractory to other forms of treatment.

[0015] The invention also includes a method of making a pharmaceutical composition comprising arsenic trioxide.

In the present invention, arsenic trioxide or melarsoprol compounds may be used alone or in combination with other known therapeutic agents (including chemotherapeutic agents, radioprotectants and radiotherapy) or in patients. It can be used in combination with techniques to improve quality of life or to treat leukemia, lymphoma or solid tumors. The arsenic compound can be used before, during, or after administration of one or more known chemotherapeutic agents, including antitumor agents. The arsenic compound can be used before, during or after radiation therapy.

[0017] The pharmaceutical compositions of the invention are sterile solutions suitable for intravenous injection or infusion. Already
In one embodiment, the present invention comprises a composition suitable for oral delivery comprising arsenic trioxide or melarsoprole and a pharmaceutically acceptable excipient or carrier.
In another embodiment, the invention also includes compositions suitable for topical or transdermal delivery, including, but not limited to, iontophoresis. The invention also provides certain treatment modalities, pharmaceutical compositions and kits.

[0018] Certain compositions of the present invention and their uses are described in the following sections and subsections.

[0019] 4. DETAILED DESCRIPTION OF THE INVENTION Methods and compositions for the treatment of leukemia, lymphoma or solid tumor are described herein. The present invention is a method of treating acute or chronic leukemia, lymphoma or solid tumor in a human, comprising administering to a human in need of such treatment a therapeutically effective non-lethal dose of one or more arsenic compounds (eg, trioxide Arsenic or melarsoprol).

The present invention also provides a method of treating leukemia in a human who has become refractory to other forms of treatment, wherein the arsenic trioxide or melarsoprol is replaced by another chemotherapeutic agent (eg, all-trans). -Retinoic acid (ATRA)) in combination with humans.

The present invention also relates to a method for producing a pharmaceutical composition comprising arsenic trioxide. The pharmaceutical compositions of the present invention preferably exhibit reduced toxicity, improved efficacy, improved stability during storage and use, and the compositions preferably have a physiologically acceptable pH. .

4.1.Arsenic compounds The “arsenic compound” used in the present invention is a pharmaceutically acceptable form of arsenic trioxide (AS)_TwoO _Three ) Or melarsoprol. Meralsoprole is synthesized by complexing melarcene oxide and dimercaprol, or purchased commercially (Rhon
e Poulenc Rorer, Arsobal® by Collegeville, PA)
Organic arsenic compound. The non-pharmaceutically formulated ingredients of the present invention are well known.
Therefore, they are manufactured by chemical techniques well known in the art.
(For example, Kirk-Othmer, Encyclopedia of Chemical Technology
 4th Edition. See Volume 3, pps. 633-655 John Wiley & Sons).

The terms “therapeutic agent”, “therapeutic regime”, “radioprotective agent”, and “chemotherapeutic agent” as used in the present invention are those skilled in the art of treating cancer, viral infections and other malignancies. Commonly known drugs and drug therapies (including vaccines). "Radiation therapy" agents are well known in the art.

In the present invention, the arsenic trioxide or melarsoprol compound is used alone or in combination with other known therapeutic agents (including chemotherapeutic agents, radioprotective agents and radiation therapy)
Alternatively, it can be used in combination with techniques for improving the quality of life of a patient or for treating leukemia, lymphoma or solid tumors. For example, the arsenic compound may include one or more known anti-tumor agents (mustard compound, nitrous mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, flox Uridine, methotrexate, vincristine, vinblastine, taxol, etoposide, temiposide, dactinomycin, daunorubicin, doxorubicin,
Including but not limited to bleomycin, mitomycin, cisplatin, carboplatin, estramustine phosphate, hydroxyurea, BCNU, procarbazine, VM-26, interferon and all-trans-retinoic acid (ATRA) or other retinoids (See, for example, Physician Desk References 1997). The arsenic compound can be used before, during or after radiation therapy.

In certain embodiments, an arsenic compound of the invention and ATRA can be administered as a mixture. In a preferred embodiment, the lymphoma, leukemia or solid tumor in a human being treated by the combination is refractory to common treatment methods or is a recurrent case of leukemia.

Any suitable method of administration including, but not limited to, parenteral administration, such as intravenous, subcutaneous, intramuscular and intrathecal administration, oral and intranasal administration, and inhalation is used in accordance with the present invention. be able to. The manner of administration will vary depending on the type of cancer and the condition of the patient.

The pharmaceutical compositions used may be in the form of sterile aqueous or organic solutions, colloidal suspensions, caplets, tablets and cachets.

4.2. Method of Treatment As used herein, the term “treatment of leukemia” means that the disease and the symptoms associated with the disease are reduced, diminished, cured, or in remission. . For example, the treatment methods of the present invention can reduce the number of white blood cells or reduce lymphocytosis in a treated human.

The term “treatment of lymphoma” as used herein means that the disease and the symptoms associated with the disease are reduced, diminished, cured, or in remission.

The term “treatment of a solid tumor” as used herein means that the disease and the symptoms associated with the solid tumor are reduced, diminished, cured, or in remission.

Further, the term “treatment of leukemic infiltration” refers to the infiltration of leukemia cells from the circulation into other organs and systems and the symptoms associated with such infiltration,
It means healing or being in remission. The term "refractory" as used herein generally means that the leukemia is resistant to treatment or cure.

As used herein, a “preneoplastic” cell refers to a cell that is in the transition from a normal type to a neoplastic type, or a cell that has failed to differentiate normally, and Morphological evidence further supported by studies indicates that preneoplasia progresses through multiple stages.

In one embodiment, the invention provides a method of treating leukemia in a human comprising administering to the human a therapeutically effective, non-lethal amount of arsenic trioxide or melarsoprole. The present invention also provides a previously undisclosed weight-based use of these compounds, which is otherwise highly toxic, to maximize safety in humans.

Arsenic trioxide (As ₂ O ₃ ) inhibits proliferation and induces apoptosis in NB4 acute promyelocytic leukemia cells. Acute promyelocytic leukemia (APL) produces a PML / RARα fusion protein between PML, a growth suppressor located on a nuclear matrix associate, and RARα, a nuclear receptor for retinoic acid (RA) With t (15; 17) translocation. PML / RARα, like dominant negative RARα mutants, has been proposed to block bone marrow differentiation through inhibition of nuclear receptor response. Furthermore, in APL cells, PML / RARα is thought to displace PML and other karyotype (NB) antigens on nuclear microplaques, resulting in loss of PML and / or NB function. It has been proposed that high concentrations of arsenic trioxide promote apoptosis, whereas low concentrations induce partial differentiation in NB4 cells and cells from APL patients. As ₂ O ₃ is alleged to act by specifically generating PML-RARα in APL cells and relocating and degrading to the nucleosome
(Zhu et al., 1997, Proc. Natl. Acad. Sci. USA, 94: 3978-3983). However, these findings tend to limit the use of arsenic trioxide to a subset of leukemias.
See Koning et al., 1997, Blood, 90: 562-570.

Surprisingly, we found that both As ₂ O ₃ and melarsoprole inhibited cell proliferation and caused apoptosis in various myeloid leukemia cell lines in a PML- and PML-RARα-independent manner. Discovered that it can be guided. Thus, contrary to previous findings, the inventors have found that arsenic trioxide and melarsoprole are both effective against a wide range of leukemias, regardless of the underlying molecular mechanism of neoplasia. I found something. Examples of the effects of arsenic compounds on many leukemic cell lines are described in Sections 5.1 and 5.2.

Therefore, the arsenic compound of the present invention can be used for various leukemias as described below, but is not limited thereto. Acute lymphoblastic leukemia (ALL) Acute lymphoblastic B-cell leukemia Acute lymphoblastic T-cell leukemia Acute myeloblastic leukemia (AML) Acute promyelocytic leukemia (APL) Acute monoblastic leukemia Acute red Leukemia leukemia Acute myeloid megakaryocytic leukemia Acute myelomonocytic leukemia Acute undifferentiated leukemia Chronic myelogenous leukemia (CML) Chronic lymphocytic leukemia (CLL) It will be understood that it is possible.

In another embodiment, the present invention provides a method of treating human lymphoma comprising administering to a human a therapeutically effective, non-lethal amount of arsenic trioxide or melarsoprol. Lymphomas that can be treated by the methods of the present invention include well-differentiated (high grade) lymphoma, intermediately differentiated (intermediate grade) lymphoma, poorly differentiated (low grade) lymphoma and various subclasses, It is not limited to these.

In yet another embodiment, the invention is directed to the treatment of a solid tumor comprising metastases in a human, comprising administering to the human a therapeutically effective and non-lethal amount of arsenic trioxide or melarsoprole. Provide the law. Solid tumors that can be treated by the methods of the present invention include, but are not limited to, gastrointestinal tract, esophagus, liver, stomach and colon, skin, brain, bone, breast, lung, and soft tissue cancers. These include, but are not limited to, various sarcomas, preferably prostate cancer.

In various embodiments, the leukemia or tumor cells infiltrate other organs and systems of the human, such as the central nervous system. The methods of the present invention can also be applied to reduce preneoplastic cell numbers in humans that have an abnormal increase in preneoplastic cell numbers.

In certain embodiments, the present invention provides a method of treating acute promyelocytic leukemia (APL) in a human comprising administering to the human a therapeutically effective, non-lethal dose of melarsoprole. provide. The inventors have discovered that concentrations of melarsoprole, which are cytotoxic in vitro, are easily achieved in vivo, as described in Section 5.2.

In one particular embodiment, the invention provides a method of treating chronic myeloid leukemia in a human, comprising administering to the human a therapeutically effective, non-lethal amount of arsenic trioxide. . The inventors have discovered that arsenic trioxide can also induce apoptosis in CML cell lines, as described in Section 5.3. The therapeutic benefits of the pharmaceutical compositions of the present invention comprising arsenic trioxide are far superior to potassium arsenite, which is usually formulated as a Fowler solution.

[0042] In yet another specific embodiment, the invention provides for the conversion of the RARα locus on chromosome 17 to chromosome 11 comprising administering to the human a therapeutically effective amount of arsenic trioxide or melarsoprole. A method of treating acute promyelocytic leukemia (APL) in humans with a locus. A
In most cases of PL, RARα on chromosome 17 translocates and fuses with the PML gene located on chromosome 15. This is described as t (15; 17). In a few cases, RAR
α translocates to chromosome 11 where it fuses with the PLZF gene. Patients with t (15; 17) have a unique sensitivity to treatment with all-trans-retinoic acid (ATRA) with a complete remission rate of 75% to 95%. APL with t (11; 17) (PLZF-RARα) has a poor response to chemotherapy, little or no response to treatment with ATRA,
This is defined as a new APL syndrome because it has a particularly poor prognosis. According to the present invention, arsenic trioxide or melarsoprol can be used to treat such cases of APL. Transgenic animal models of APL with t (15; 17) and t (11; 17) for testing the therapeutic benefit and dosage of the arsenic compounds of the invention are described in Section 5.4 below.

[0043] Humans with leukemia may be refractory to conventional treatments because they have undergone anti-leukemia treatment (eg, chemotherapy). Thus, the present invention provides a therapeutically effective non-lethal amount of an arsenic compound, and another chemotherapeutic agent,
For example, a method of treating leukemia in a human that is unresponsive to conventional treatments, including, but not limited to, administering to a human a combination of all-trans-retinoic acid (ATRA) or other retinoids. provide. The arsenic compound may be arsenic trioxide or melarsoprol or any of its pharmaceutically acceptable salts. The invention also includes treating a retinoid-resistant patient with an arsenic compound.

In certain embodiments, the arsenic compound of the invention and the chemotherapeutic agent may be administered as a mixture or continuously. If administered continuously, the arsenic compound can be administered either before or after the chemotherapeutic agent, as long as the first drug is administered while the second drug is administered while having anti-leukemic activity in humans. You may. Any of the administration methods described herein may be used to deliver the above combinations. In a preferred embodiment, the human leukemia treated by the combination is
Cases that are refractory to common therapies or that have recurred leukemia.

4.3. Method for Producing a Sterile Arsenic Trioxide Solution The arsenic compounds of the present invention are formulated into sterile pharmaceutical preparations for administration to humans for the treatment of leukemia, lymphoma, and solid tumors. be able to. A composition comprising a compound of the present invention formulated in a usable pharmaceutical carrier is prepared, packaged, and labeled for treatment of an indication of leukemia, lymphoma, or solid tumor, and treated accordingly. Can be used for

In one aspect, the invention provides a method of making a pharmaceutical composition comprising a therapeutically effective, non-lethal amount of arsenic trioxide (As ₂ O ₃ ). Arsenic trioxide (raw material) is a solid inorganic compound and is commercially available in a very high purity form. However, it is difficult to dissolve arsenic trioxide in an aqueous solution. In addition, there are no published reports on how to formulate arsenic trioxide as a pharmaceutical composition suitable for direct injection into humans. Arsenic exists in +5 valence state (pentavalent) or +3 valence state (trivalent) in solution. For example, potassium arsenite (KAsO ₂ ; it is present in Fowler's solution) and salts of arsenous acid contain pentavalent arsenic. It is known that one of them is more toxic than the other. (The Pharmacological Basis of Theraepu by Goodman & Gilman
tics, 9th edition, Chapter 66, 1660, 1997). A fresh solution of arsenic trioxide containing trivalent arsenic is gradually oxidized to pentavalent when exposed to air for a long time, and the relative toxicity of the As ₂ O ₃ solution is increased by the accumulation of pentavalent arsenic. Changes over time. (Id.) In addition, it is observed that the total amount of arsenic in the solution decreases with time. The phenomenon of loss of this substance occurs as arsenic in solution is gradually converted to arsenic hydride (AsH ₃ ), a gaseous compound at room temperature. This is particularly problematic in pharmaceutical applications if the concentration of the active ingredient in the injectables cannot be controlled. In addition, arsenic hydride is also toxic and it is undesirable to escape from solution into the surrounding air.

The present inventors have conducted experiments and have successfully developed a method for formulating arsenic trioxide that overcomes the above-mentioned solubility and stability problems. The method is based on the high purity of solids in aqueous solutions.
Including solubilizing As ₂ O ₃ at high pH such that the pH is above 12. For example, a 5 M aqueous sodium hydroxide solution can be used. Mechanical agitation and / or mild heating can be applied to aid solubilization and obtain a clear and homogeneous solution. As ₂ O ₃ solutions can also be obtained by dissolving solid compounds overnight. Typically, a 1 M As ₂ O ₃ solution is obtained by this method. However, this solution is too basic for a pharmaceutical composition.

To adjust the pH of the As ₂ O ₃ solution, first dilute the solution with water, eg, about 1 mg
/ mL, pH12. The As ₂ O ₃ solution is back titrated with an acid such as hydrochloric acid (1M to 5M HCl) with constant stirring until the pH is about 8.0 to 8.5. High concentrations of hydrochloric acid are not appropriate because they cause precipitation in the As ₂ O ₃ solution. The partially neutralized As ₂ O ₃ solution is then made sterile, for example by filtration (eg, through a 0.22 μm filter), and stored in a sterile vial.

To make a pharmaceutical composition that can be injected directly into a subject, the composition must be sterile
Rather, standard aseptic techniques known to those skilled in the art can be used. For example,
See Remington's Pharmaceutical Science. This partially neutralized As_TwoO _Three The solution can be further adjusted to near physiological pH by diluting (10-100 times) with a pharmaceutical carrier such as a 5% glucose solution. For example, 10 mL of partially
Neutralized As_TwoO_ThreeAdd the solution into 500 mL of 5% glucose solution to bring the final pH to about 6.5 to 7
.5. This method of the present invention reduces the oxidation of arsenic in solution. Book
Pharmaceutical composition containing arsenic trioxide produced by the method of the invention has improved stability and long-term storage
Indicated that it is possible.

4.4 Pharmaceutical Compositions and Methods of Administration In accordance with the present invention, arsenic compounds and physiologically acceptable solvents can be formulated for oral or parenteral administration.

For oral administration, the pharmaceutical preparation can be in liquid form, eg, a solution, syrup, or suspension, or can be reconstituted with water or other suitable vehicle before reconstitution with a pharmaceutical preparation. can do. Such liquid formulations may contain pharmaceutically acceptable excipients such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fat), emulsifiers (e.g., lecithin or acacia), non-aqueous vehicles (e.g., It can be prepared in a conventional manner using almond oil, oily esters, or fractionated vegetable oils), and preservatives (eg, methyl or propyl-p-hydroxybenzoic acid or sorbic acid). Pharmaceutical compositions include, for example, pharmaceutically acceptable excipients, such as a binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), a bulking agent (e.g., lactose, microcrystalline cellulose, or phosphoric acid). Traditionally using calcium hydrogen), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate) It can also be in the form of tablets or capsules prepared by the method. Tablets can be coated by methods well known in the art.

For administration by inhalation, the compounds used according to the invention are treated with a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from pressurized packs or nebulizers. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules or cartridges for use in an inhaler or insufflator, for example those made of gelatin, may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. The dosage form for injection is, for example, a unit dosage form such as an ampoule or a container for multiple administrations, and can be formulated by adding a preservative.
The pharmaceutical compositions may be in the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The present invention also provides kits for performing the therapeutic regimes of the present invention.
Such kits consist of one or more containers containing a therapeutically effective amount of an arsenic compound in a pharmaceutically acceptable dosage form. The arsenic compound in one vial of the kit of the invention is combined in a pharmaceutically acceptable solution, for example, with a sterile saline solution, a glucose solution, or a buffer solution, or other pharmaceutically acceptable sterile liquid. It can be shaped. Alternatively, the complex can be lyophilized or dried; in this case, the kit optionally further comprises a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.) in a container, preferably a sterile solution. The complex can then be reconstituted to make a solution for injection.

In another embodiment, the kit of the present invention further comprises a needle or syringe, preferably sterilely packaged, for injecting the conjugate, and / or a prepackaged alcohol pad. Consists of Instructions for use of the arsenic compound by a physician or patient can optionally be included.

The degree of therapeutic dose of an arsenic compound in the treatment of acute or chronic leukemia will vary depending on the severity of the condition to be treated and the route of administration. Dosage, and perhaps frequency, will also vary depending on the age, weight, condition, and response to treatment of the individual patient. Usually, the daily dose range of arsenic trioxide for the conditions described herein is from about 0.05 to about 5 mg / kg body weight, divided into several injections, or orally,
Or administer locally. A preferred total daily dose is from about 2.5 to about 40 as arsenic trioxide
mg. Preferably, the arsenic trioxide agent of the invention is administered daily, for up to 60 days, or until remission is observed, followed by 2 to 10 cycles, each cycle lasting about 25 days. For example, depending on the weight of the patient with acute promyelocytic leukemia, a daily dose of arsenic trioxide of 10 mg or more can be administered. Alternatively, a therapeutic effect may be obtained with a daily dose of less than 10 mg of arsenic trioxide according to a body weight-based dosage regime.

For the treatment of solid tumors, a preferred dosage regimen includes intravenous infusion of about 0.1 to about 5 mg / kg body weight per day for 5 days. This 5-day treatment protocol is repeated once a month until tumor growth is inhibited or the tumor shows signs of regression.

With respect to melarsoprol, the total daily dosage for the conditions described herein usually is about 0.1 to about 5 mg / kg body weight divided into several parenteral,
Oral or topical administration. A preferred total daily dose is from about 0.5 to about 4 mg melarsoprol / kg body weight.

The effectiveness of arsenic trioxide or melarsoprole therapy on the development and progression of cancer can be monitored by any method known in the art, including, for example, carcinoembryonic antigen (CEA), α -Levels of tumor-specific antigens and putative biomarkers, such as fetoprotein, and measurements of morphological and / or size changes using CT scans and / or ultrasound, including but not limited to.

The desired blood concentration can be maintained by continuous infusion of the arsenic compound while confirming the plasma concentration. The attending physician does not know how and when to terminate, discontinue, or adjust therapy to lower doses due to toxicity or damage to the bone marrow, liver, or kidneys. It should be noted. Conversely, the attending physician may not have a sufficient clinical response (except for toxic side effects).
It also knows how and when to adjust the therapy to possibly higher doses.

Furthermore, any suitable route of administration may be employed for providing a patient with an effective amount of an arsenic compound. For example, oral, transdermal, iontophoretic, parenteral (subcutaneous, intramuscular, intrathecal, etc.) can be performed. Dosage forms include tablets, troches, cachets, dispersions, suspensions, solutions, capsules, patches, and the like. (See Remington's Pharmaceutical Sciences).

The pharmaceutical composition of the present invention comprises an arsenic compound as an active ingredient, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients such as all-trans-retinoic acid. Can be contained. "Pharmaceutically acceptable salts" means salts prepared with pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.

Pharmaceutical compositions include those suitable for oral, mucosal route, transdermal, iontophoretic, injection (including subcutaneous, intramuscular, intrathecal, and intravenous) administration, but given The most suitable route of administration for an example will depend on the nature and severity of the condition being treated.

In the case of intravenous injection or infusion of the composition, suitable dose ranges for use include, for example, about 1 to about 40 mg of arsenic trioxide per day, total Dosage is about 0.001 to about 10 mg arsenic trioxide / kg body weight, or a total daily dose of about 0.1 to about 10 mg melarsoprole / kg body weight.

In addition, arsenic carriers can be delivered via charged and uncharged matrices used as drug delivery systems, such as cellulose acetate membranes, and targeted delivery, such as fusogenic liposomes, attached to antibodies or specific antigens. Can be delivered via the system.

In practical use, the arsenic compounds can be combined as the active ingredient in making intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a variety of forms depending on the form of administration required, for example, oral or by injection (including tablets, capsules, powders, intravenous injection or infusion). Pharmaceutical solvents commonly used to prepare compositions for oral dosage form include, for example, water, glycols, oils, fats, alcohols, flavoring agents, preservatives, coloring agents, and the like, in any form. In the case of oral liquid preparations, for example, suspensions, solutions, elixirs, liposomes and aerosols; starch, sugars,
Microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants and the like, or powders, capsules and tablets in the case of oral solid preparations can be used. In preparing injectable forms, for example, compositions for intravenous injection or infusion, similar pharmaceutical media include, for example, water, glycols, oils, buffers, sugars,
Preservatives and the like can be used as are known to those skilled in the art. Such injectable compositions include, but are not limited to, 5% w / v dextrose, saline, or other solutions. The total dose of the arsenic compound can be administered in one vial of intravenous liquid, for example, in the range of about 2 mL to about 2000 mL. The volume of the dilution depends on the total dose to be administered. For example, arsenic trioxide supplied in 10 mL of an aqueous solution at a concentration of 1 mg / mL is diluted with 10 to 500 mL of a 5% glucose solution and intravenously over a period ranging from about 10 minutes to about 4 hours. Be infused.

As an example of a course of treatment for patients with leukemia, lymphoma, or solid tumor, intravenous infusion of an arsenic trioxide aqueous solution is administered daily, and about 0.01 to 1 mg of arsenic trioxide per kg of patient weight is administered as a daily dose. can do. Preferably, a dose of about 0.15 mg arsenic trioxide per kg body weight per day is used. This course of treatment can be continued until remission of the bone marrow is observed or until the adverse effects become severe. This treatment course is approximately 10 with 3 to 6 weeks between each course
Can be repeated up to 10 times over a month. As a course of treatment after remission, arsenic trioxide is infused as a daily dose of about 0.15 mg per kg of patient's body weight for 25 days in a daily or weekday only manner.

5. Examples The following are examples of the use of the arsenic compounds of the present invention in the treatment of various leukemias. These and other experiments indicated that the arsenic compound dosage forms of the present invention were well tolerated in humans. For example, three APL patients were intravenously injected (at the same dose) once daily with 10 mg of the arsenic trioxide preparation of the present invention.

5.1. Arsenic trioxide and melarsoprole induce apoptosis in myeloid leukemia cell line As ₂ O ₃ and melarsoprole are retinoic acid-resistant cell lines derived from APL cell line NB4-306 (NB4 The activity of the native PML-RARα fusion protein is no longer expressed), HL-60, KG-1, and myeloid leukemia cell lines including myeloid monocytic cell line U937. To investigate the role of PML in mediating the activity of arsenic compounds, we used these agents to convert mouse embryonic fibroblasts (MEFs) in which the PML gene was inactivated in cells by homologous recombination. And bone marrow (BM) progenitor cells. Unexpectedly, both compounds inhibited cell proliferation and induced apoptosis in all of the cell lines tested. Meralsoprole is 10 ^-7 to 10 ^-8 mol
In equimolar concentrations in the range of / L was more potent than As ₂ O _3. As ₂ O ₃ is PML and PML-RARα
Is relocalized on the karyotype, followed by degradation of PML in NB4 and HL60 and U937 cell lines. Meralsoprole was more potent in inhibiting proliferation and inducing apoptosis, but did not affect nuclear localization of PML and / or PML-RARα. Moreover, both As ₂ O ₃ and melarsoprole inhibit the growth of PML + / + and PML − / − MEF and induce apoptosis to the same extent, and
+ / + And PML − / − progenitor cells in CBM-E (colony forming unit eryth
roid) and the formation of CFU-GM (colony forming unit granulocyte-monocyte). Detailed descriptions of these methods and materials and the results of these experiments are provided by Wang et al.
, Blood, 1998, 92: 1497-1504.

The results of these experiments indicate that the cytotoxic effects of the two arsenic compounds in these cell lines are not mediated by a mechanism of action that depends on PML or PML-RARα expression. In most cell lines, melarsoprol is somewhat potent in inhibition and induction of apoptosis compared Then cell proliferation and As ₂ O _3, the efficacy of both drugs were dose dependent. As previously reported, As ₂ O _3, while triggering apoptosis, the PML protein in NB4 cells were re-localized on karyoplast, has been confirmed to induce the degradation of PML and PML-RAR [alpha ing. However, similar potency has been observed in HL60 and U937 cells without the PML-RARα fusion gene. In addition, melarsoprole was expressed in all cell lines tested in PML and / or PML-
Apoptosis was induced without changing RARα.

The differentiation effects of As ₂ O ₃ and melarsoprol appeared to be negligible in vitro and may be dependent on the expression and / or modification of either PML and / or PML-RARα. Did not. In fact, the small effects we observed during long-term (up to 2 weeks) culture were comparable in all cell lines tested with both compounds.

Also, down-regulation of bcl-2, which has long been associated with the anti-leukemic effect of As ₂ O ₃ on APL, has been linked to the natural protein (P
ML-RARα) has also been found to be independent of PML-RARα protein expression, as it also occurs in NB4 subclone 306 where it is not detected. Finally, to determine whether PML expression is essential for the anti-leukemic effect of arsenic compounds,
PML was examined in embryonic fibroblasts and bone marrow cells from mice in which homologous recombination had been eliminated. In these cells lacking any expression of PML, both of As ₂ O ₃ and melarsoprol are effective as the growth inhibition and apoptosis induction, both colonies normal CFU-E and CFU-GM Similar effects on formation were shown. Furthermore, no difference was observed between wild type and PML − / − cells. Without being limited by any theory, these data strongly support the theory that the anti-leukemic effects of these arsenic compounds occur independently of the expression of both PML and PML-RARα. These results are consistent with the pharmaceutical history that arsenic compounds have been used in diseases not characterized by alterations in PML proteins, such as chronic myeloid leukemia.

These results indicate that both As ₂ O ₃ and melarsoprol are widely effective as anti-leukemic agents in both myeloid and lymphocytic disorders. In conclusion, these data indicate that cytotoxic activity is not mediated by PML proteins and, therefore, is not limited to diseases associated with altered PML expression. Thus, the arsenic compounds of the present invention may have a broader therapeutic role without being limited to APL.

5.2. Clinical trial of melarsoprole in patients with advanced leukemia Meralsoprole, an organic arsenical synthesized by combining melarcene oxide with dimercaprol, has been mainly used for the treatment of African trypanosomiasis.
The effect of melarsoprol on the induction of apoptosis in cell lines showing chronic B-cell lymphoproliferative disorders has been studied, the results of which are described below.

[0075] Meralsoprol (Rhone Poulenc Rorer; Arsobal [36 mg / mL] supplied by Collegeville, PA) was used at a raw material concentration of 10 ^-4 mol / L.
And stored at room temperature. As ₂ O ₃ (Sigma, St. Louis, Mo.) was dissolved in 1.65 mol / L sodium hydroxide (NaOH) at a raw material solution of 10 ⁻³ mol / L. It was serially diluted (10 ^-6 to 10 ^-9 mol / L) with RPMI1640 medium. Epstein-Barr virus (EBV) transformed B prolymphoid cell line (JVM-2), EBV transformed B cell chronic lymphocytic leukemia (B-CLL)
) A cell line (I83CLL) as well as one non-EBV transformed B-CLL cell line (WSU-CLL) were used as targets. Melarsoprol (10 ^- 10 ^- mol /
L) was performed over 96 hours.

Unexpectedly, the inventors have found that melarsoprole causes a dose- and time-dependent inhibition of survival and growth in all three cell lines. In contrast, similar concentrations of As ₂ O ₃ had no effect on either viability or growth. After 24 hours, all three cell lines treated with melarsoprole (10 ^- mol / L) exhibited morphological features of apoptosis. WSU-C
Significant concentration-dependent down-regulation of bcl-2 mRNA was observed after exposure of LL 183CLL, as well as JVM-2 cells, to melarsoprole for 24 hours. Furthermore, all three cell lines, whereas was also observed a decrease in bcl-2 protein expression, As ₂ O ₃ did not have any effect in this parameter.

It is hypothesized that the above in vitro data unexpectedly demonstrates the broad anti-leukemic effect of melarsoprol on both myeloid and lymphoid cells, and generally at concentrations lower than As ₂ O _3. The study was then initiated to evaluate the pharmacokinetics, safety, and potential efficacy of melarsoprol in patients with relapsed or refractory leukemia.

Eligible patients received the following treatments: A short IV injection was performed once a day for 3 days, which was repeated weekly for 3 weeks. Patients who responded received the same treatment for another 3 weeks. The initial dose was 1 mg / kg on day 1, 2 mg / kg on day 2,
On day 3, the dose was 3.6 mg / kg, and on all subsequent days the dose was the same as on day 3. As parallel in vitro tests, culture sensitivity of fresh leukemia cells to both melarsoprole and As ₂ O ₃ and continuous flow cytometric measurements of surface antigen expression, apoptosis, and bcl-2 expression were performed. Three patients with AML and one patient with CML participated in this study.

Using a high performance liquid chromatography-based method sensitive to about 10 mg / ml, preliminary pharmacokinetic data indicate that the Cm from 1.2 ng / ml on day 1 to 2.4 ng / ml on day 3
It can be seen that the maximum plasma drug concentration is obtained immediately after injection of ax. The initial distribution period was acute, while prolonged T1 / 2γ indicated release from the deep compartment. The plasma area (AUC) under the concentration x time curve was 0.48 ng · h / ml on the first day.
From day 3 to 1.48 ng · h / ml. Detectable concentrations of the drug were found in plasma one week after the first dose. The drug was relatively well tolerated. The adverse effects included temporary pain at the injection site and mild nausea. There were no signs of "reactive brain disease" (possibly found during treatment of CNS trypanosomiasis).

From the results of these studies, it can be seen that melarsoprol may have a broader effect than inorganic As ₂ O _{3 and} that it is cytotoxic to leukemic cells in vitro,
Thus, it can be seen that therapeutically effective concentrations are easily achieved in vivo.

5.3. Arsenic trioxide can be expressed in arsenic trioxide (A) using the Philadelphia chromosome positive CML cell line K562, which induces apoptosis in K562 chronic myeloid leukemia (CML) cells.
s ₂ O ₃ ) to promote apoptosis in CML. The suspension culture cells in the logarithmic growth phase, concentration and exposed to As ₂ O _3, respectively 1x10 ^-5 M, 5x10 ^-6 M, and 1x10 ^-6 M. Aliquots of cells were analyzed at various time points during the 72 hour process to assess viability and apoptosis. Viability was measured using trypan blue exclusion. At the same time, apoptosis was detected by morphology, flow cytometry, and DNA gel electrophoresis.

Arsenic trioxide at a concentration of 1 × 10 ⁻⁶ M had no effect on the growth or viability of K562 cells. The greatest effect on cell growth and viability is 1 × 10 ⁻⁵ M
In As ₂ O ₃ . The K562 cell growth and viability data after exposure to As ₂ O ₃ are recorded in Table 1 over 72 hours.

[0083]

[Table 1]

The evidence that this arsenic-induced decrease in viability was indicative of apoptosis was analyzed.
Ten^-FiveM's As_TwoO_ThreeStained cells of K562 cells incubated for 72 hours
Morphological features of apoptosis, such as membrane blebbing and nuclear condensation, in the cytospin
Was evident. This is 10^-FiveM's As_TwoO_ThreeExtracted from K562 cells exposed to
Of DNA intranucleosome damage visualized by gel electrophoresis of isolated DNA
Correlated with traces. Quantitative evaluation of apoptosis as measured by the TUNEL method showed that 75.6% ± 8.6 (1 × 10 5^-FiveM
As_TwoO_Three) Cells were found to exhibit apoptosis. Ten^-FiveM's As _Two O_ThreeTreatment of K562 cells with p21 detected by Northern analysis
 Up-regulation of mRNA occurred, implying cell arrest in the G1 phase of the cell cycle. This data is for therapeutic drugs for CML
As arsenic trioxide as the.

5.4. PML-RAR [alpha and PLZF-RAR [alpha transgenic mice definitive retinoic acid and arsenic trioxide (As ₂ O ₃₎ study acute promyelocytic leukemia using (APL) is associated with chromosomal translocations, this translocation , 17
Other loci in the genome of the retinoic acid receptor α (RARα) locus on chromosome (eg, the PML gene located on chromosome 15 in most APL cases,
And, rarely, it always involves translocation to chromosome 11 (PLZF gene). t (15;
Patients with 17) respond to treatment with all-trans retinoic acid (ATRA) with a complete remission rate of 75-95%. APL with t (11; 17) (PLZF
-RARa) has a low response to ATRA.

[0086] To test the efficacy of As ₂ O _{3 in} treating APL, a disease model was created in transgenic mice. The transgenic mice underwent PML-RARa under control of the myeloid-promyelocytic specific human cathepsin G (hCG) minigene.
Alternatively, it was prepared by a standard technique for expressing a PLZF / RARa fusion protein. Both hCG-PML-RARa and hCG-PLZF-RARa transgenic mice developed myeloid leukemia with APL characteristics similar to humans.

A trial of the above leukemic mice was started using the following treatment regime. 1) ATRA:
Oral administration of 1.5 μg / g body weight per day; 2) ATRA: Intraperitoneal administration of 6 μg / g body weight per day. Mice were bled weekly to assess response.

PML / RARα leukemia responded well to ATRA and had a high remission rate (80% with treatment plan 1). Surprisingly, in vitro, ATRA is not compatible with PML-RA
In bone marrow of both Rα and PLZF-RARa leukemia, and in the spleen precursor assay, it induced differentiation and inhibited leukemic cell proliferation and leukemia colony formation. Furthermore, in ex vivo experiments, when leukemia cells from PLZF-RARa mice were pre-incubated with ATRA and transplanted into receptor nude mice, the leukemia cells lost tumorigenicity, whereas untreated Cells were tumorigenic. However, in vivo, PLZF-RARa leukemia had a low response to ATRA (28% with treatment plan 1), but with increasing ATRA dose,
The effect increased (50% in treatment plan 2). In conclusion, leukemia in PLZF-RARa transgenic mice is responsive to ATRA treatment but has a high A
Treatment with a TRA dose may be required. These test results indicate that t
(11; 17) has direct implications for the treatment of APL patients.

In both PML-RARa and PLZF-RARa leukemia, ATR
A prolongs survival, but shortly after remission, the leukemia recurs,
Refractory to continued ATRA treatment. Furthermore, using two transgenic mice models, APL patients ATRA resistant, as well as t (11; 17) for the treatment of APL with, As ₂ O ₃ alone, and in combination of the efficacy of ATRA + As ₂ O ₃ As well as dose. A dose of 6 μg of As ₂ O ₃ per day, or a combined dose of 6 μg of As ₂ O ₃ and 1.5 or 6 μg of ATRA per g of body weight per day is administered intraperitoneally. Mice are bled weekly to assess APL remission.

5.5. Manufacturing and Safety of Pharmaceutical Formulations Solid ultra pure arsenic trioxide (As ₂ O ₃ ) was converted to 5M sodium hydroxide (NaOH).
Dissolved in the solution. The suspension was stirred at room temperature for 5 minutes, resulting in a clear, homogeneous solution. This As ₂ O ₃ solution (2 mL, 1.0 M) was added to 393.6 mL of H ₂ O in a 500 mL Erlenmeyer flask. At this time, the concentration of As ₂ O ₃ is 1 at pH = 12.
mg / mL. Dilute HCl (49.26 mL, 37% wt / wt, 10/15 M) with H ₂ O (50.74 mL) in a 250 mL Erlenmeyer flask to give 5.0 M H ₂ O.
A Cl solution was prepared. The HCl solution was then transferred using a syringe to an evacuated, empty 1000 mL container. The As ₂ O ₃ solution was back titrated with HCl (0.725 mL, 5.0 M) to pH 8.0. About 10 mL of the back titrated As ₂ O ₃ solution was filtered using a Millex GS 0.22 μm filter device and added to each of about 30 sterilized and evacuated sterile vials. To prepare a pharmaceutical formulation to be administered intraperitoneally to a patient, 10 mL of the above solution was removed from two vials and added to 500 mL of a 5% dextrose solution. Incidentally, the final pH of this solution was 6.5.

Atomic absorption analysis confirmed the high purity of the bulk starting material (see Table 1). Duplicate samples of the four intermediate, or final, solutions were assayed for total arsenic content. The assay bulk powder confirmed the very high purity of the starting material. The arsenic content data of the intermediate and final product solutions are shown in Table 2 below.

The data shown below show that the solution is stable, as there are no signs of arsenic weight loss over time.

[0093]

[Table 2]

6. Example: Clinical Trial of APL Patients In APL patients, arsenic trioxide was evaluated to determine whether the drug induced cell differentiation or apoptosis. 12 relapsed from various previous treatments
One patient was treated with a daily dose of 0.06-0.2 mg / kg arsenic trioxide until remission of the bone marrow was achieved. Flow cytometry for immunophenotype, fluorescence
By in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR) assay for PML / RARa expression, and Western blot expression of apoptosis-associated proteins, caspases 1, 2 and 3
Bone marrow mononuclear cells were monitored continuously. From these results, a low dose of arsenic trioxide
It has been shown to be very effective in inducing a complete remission in patients with APL relapse. The clinical response is associated with defective cell differentiation in leukemic cells, as well as induction of apoptosis by caspase activation.

6.1. Methods Clinical Protocol: Eligibility requirements include cytogenetics or fluorescence in situ hybridization (FISH) analysis for t (15; 17) translocation, or reverse transcriptase polymerase reaction (RTT) for PML / RARa. APL-confirmed APL diagnosis was included. Patients are limited to those who have relapsed from standard therapy with all-trans retinoic acid plus a cytotoxic drug. Require signed informed consent. In addition, the organization review committee of this center,
The protocol was reviewed and approved.

Arsenic Trioxide Treatment: Arsenic trioxide was supplied as an aqueous solution in a 10 ml vial containing 1 mg / ml drug. The drug was further diluted with 500 ml of a 5% dextrose solution and injected intravenously once daily for 2-4 hours. The first patient cohort received a uniform dose of either 10 or 15 mg / day, but was given by two children referred to a previously unknown weight-based treatment regimen (0.15 mg / day). kg / day) of the present invention. Drugs were administered daily until bone marrow remission was observed. Patients who achieved a complete remission were eligible for treatment on an additional course of treatment 3-6 weeks after the earlier course. Subsequent courses are generally 0.15 mg / kg / kg for a total of 25 days, either on a daily or weekday only schedule.
It was administered at a daily dose of up to a total of 6 courses over about 10 months.

Monitoring during the study: Patients with coagulation disorders are infused with platelets and fresh frozen plasma,
Platelet counts and fibrinogen were measured at target levels ≧ 50,000 cells / cu mm and ≧ 100 mg / cu
Each was maintained at dL. Blood count, coagulation test, serum chemistry profile, urinalysis,
In addition, electrocardiograms were obtained continuously. Bone marrow aspiration and / or biopsy was performed at baseline and then periodically until remission was recorded. Normal response criteria were observed, including bone marrow ≤5% blasts, peripheral blood leukocytes ≥3,000 cells / cu.
mm, as well as recovery of platelets ≧ 100,000 cells / cu mm.

Cellular immunophenotype test: Heparinized bone marrow or blood samples were collected and mononuclear cells were isolated by Ficoll-Hypaque centrifugation. Direct immunofluorescent staining using fluorescein isothiocyanate (FITC) or phycoerythrin-binding monoclonal antibodies: CD16 (Leu 11a), CD11b, CD33 (Leu M9), HLA-DR, CD45, and CD14 (Bect
on-Dickinson (Mountain View, CA) or Immunotech Immunol
ogy (purchased from either Marseille, France)) to detect surface membrane antigens. At the same time, two monoclonal antibodies (CD33-PE / CD11b-FIT
C and CD33-PE / CD16-FITC) were used to perform the two-color staining by incubating the cells. Negative controls using irrelevant monoclonal immunoglobulins of the same isotype were analyzed simultaneously. EPICS Profile II flow cytometer equipped with a 488 nm argon laser (Coulter Elec
tronics), flow cytometry analysis was performed. Anterior and lateral scatter cell parameters were measured to identify the population of interest in conjunction with CD45 / CD14 staining and to exclude mononuclear cells from the analysis gate. Data acquisition and analysis were performed using a multi-parameter data acquisition and display system (MDADS, Coulter Electronics).

[0099] Fluorescence in situ hybridization (FISH): For a selected specimen immunofluorescently stained for CD33 and CD11b, cells that co-expressed both antigens were separated using a FACStar Plus cell sorter (Becton-Dickinson). The separated cells were incubated in a medium at 37 ° C. for 1 hour, treated with a hypotonic solution 0.075 M KCl for 5 minutes, fixed in a 3: 1 methanol: acetic acid fixative, and air-dried. Interphase FISH was performed using a specific PML / RARa translocation two-color probe (Vysis; Downers Grove, IL). Briefly, DNA from interphase cells was denatured by immersing slides in a 50% formamide / 2 × SSC solution at 73 ° C. for 5 minutes, then slides were dehydrated with alcohol and air dried. The probe mixture in the hybridization mixture was used, covered with a coverslip and sealed with rubber cement. Hybridization was performed at 37 ° C. in a humid chamber for about 12-16 hours. Following hybridization, the slides were washed three times for 10 minutes each at 45 ° C. with a 50% formamide / 2 × SSC solution, followed by an additional 5 minutes at 45 ° C. with a 2 × SSC / 0.1 NP-40 solution. Unbound probe was removed. The slides were then air dried, counterstained with 4 ', 6-diamidino-2-phenylindole and covered with a glass cover slip. Photometry attached to Zeiss Axioscope (Photoemetrics
) The fluorescent signal of the interphase cells was analyzed with a Sensys camera. At least 300 cells were analyzed per sample.

Western blot analysis: 50 mM Tris-HCl, 0.5 mM ethylene glycol [bis]-[aminioacyl] tetraacetic acid, 170 mM NaCl, 1 mM dithiothreitol, 0.2% NP-40, 0.01 U / mL aprotinin, 10 μg / mL leupeptin , 10 μg / mL pepstatin, and 1 μM phenylmethylsulfonyl fluoride (
Cells were lysed in a buffer containing (all from Sigma). The lysate was then sonicated using an ultrasonic homogenizer (471C series, Cole Parmer Instruments; Chicago, Ill.) And centrifuged at 7,500 g (Sorvall I).
nstruments; Newton, CT). The protein content of the lysate was determined using a BioRad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) at 595 nm BSA standard. 10% glycerol, 0.4% SDS, 0.1% in 1x enrichment buffer (Tris base 0.5M, 0.8% SDS).
A sample buffer containing 3% bromophenol blue, 0.2% pyronin Y and 20% 2-mercaptoethanol was added to the cell lysate and heat denatured at 95 ° C for 3 minutes. Next, 15 μg / protein lane was loaded on an SDS-polyacrylamide gel containing 12.5% polyacrylamide and size fractionated by electrophoresis. Proteins were electroblotted onto Tras-Blot® transfer medium (Bio-Rad) and stained with Ponceau-S as an internal loading control. A rabbit anti-human monoclonal antibody containing caspase 1, caspase 2 (both from Santa Cruz Biotechnology; Santa Cruz, CA), and caspase 3 (PharMingen; San Diego, CA) was added and an ECL ^™ chemiluminescence detector (Amersham; Bound antibodies were detected using Arlington Heights, Ill.). Protein bands were quantified by computer densimetry.

RT-PCR analysis of PML / RARα: RT-PCR
(Miller et al., 1992, Proc. Natl. Acad. Sci. 89: 2694-8; Miller et al., 1993, Blood 82: 1689-94).

6.2. Results Patients: Twelve patients with relapsed or refractory APL were treated. All patients had extensive prior treatment with retinoids and cytotoxic drugs (Table 3). Two patients relapsed from the allogeneic bone marrow transplant and one failed to reinject the donor T cells. One patient continued on hemodialysis due to chronic renal failure.

Clinical effects: Eleven of the twelve patients had a complete remission after arsenic trioxide treatment. Hemodialysis patients had intracranial hemorrhage on day 1 and died on day 5. The median duration of treatment for patients who responded was 33 days (range, 12-39 days), the median daily dose was 0.16 mg / kg (range, 0.06-0.2 mg / kg) and induced The median cumulative dose during the period was 360 mg (range, 160-515 mg) (Table 3). Complete remissions for all criteria were obtained when the median number of days after initiation of treatment was 47 days (range, 24-83 days). Remission by bone marrow criteria-factors that determine whether to discontinue treatment-was first obtained, usually followed by recovery of peripheral blood leukocytes and platelets. No differences in efficacy or response time were apparent over the entire dose range used in this study. After two courses of treatment, eight of the 11 patients tested had a positive to negative RT-PCR assay for PML / RAR-α.

All 11 patients in complete remission received at least one course of post-remission treatment with arsenic trioxide. Four, two, and one patients received a total of 3, 4, and 5 treatments, respectively. The median duration of remission is 5+ months (range, 1-9 + months). However, three of the eleven patients
Recurred during the second course of treatment. Since the results of the RT-PCR assay did not change in any of these patients, it was speculated that each of them had rapidly acquired drug resistance.
Later, two of these patients died due to progressive leukemia.

Adverse Events: The clinical status of the patients in this study was very diverse. It reflected a wide range of previous treatments. Because the protocol did not require hospitalization, three patients completed the guided treatment completely as outpatients, but only one other patient was hospitalized to have an intravenous catheter. However, eight patients were hospitalized for complications of leukemia. Five of these were moved to intensive care units, tracheal intubation, and due to complications such as pulmonary hemorrhage, renal failure, sepsis, graft-versus-host disease, nonspecific lung infiltration, or hypotension. Assisted breathing was required. One patient required the insertion of a permanent pacemaker after the appearance of a second-degree heart block in the setting of severe metabolic acidosis, hyperkalemia, hypotension, and renal failure. However, despite re-challenge with additional arsenic trioxide therapy, the heart block turned better. For five patients, medication was temporarily discontinued for a median of two days (range, 1-5 days) due to the appearance of severe intervening complications. Two patients had symptoms similar to those of "retinoic acid syndrome." Based on estimates, treatment of the two patients with dexamethasone improved their symptoms. Only two patients did not require any platelet transfusions. The median number of platelet units transfused was 61 (range, 0-586 units).

The median total peripheral blood lymphocyte count at the start was 4,700 cells / mm ³ (range, 500-144,000 cells / mm ³ ). Six patients developed leukocytosis (ie, ≧ 20,000 cells / mm ³ ), with a range of 20,800-144,200 cells / mm ³ . No additional treatment was given to these patients, but all patients recovered from leukocytosis without further intervention.

Common adverse reactions included dizziness during transfusion, fatigue, musculoskeletal pain, and mild hyperglycemia. Allodynia appeared in three patients, probably due to peripheral nerve disease. However, two of these patients had been stationary for long periods of time during assisted breathing, and the other patients had a natural history of neurological disease.

Immunophenotyping studies: APL is characterized by cells expressing CD33. CD3
3 is an antigen typically associated with primordial myeloid cells. Arsenic trioxide therapy gradually reduced the percentage of cells expressing CD33 alone and increased the percentage of cells expressing CD11b, an antigen associated with mature myeloid elements. Although these changes were expected from any agent that elicited remission of APL, arsenic trioxide also induced expression of cells that express both antigens simultaneously. In most cases, these dual expressing cells make up a significant portion of the myeloid cells, and this condition
After a complete remission was obtained by clinical criteria, it persisted for an extended period.

Fluorescence in situ hybridization analysis: Bone marrow mononuclear cells collected from patients early and late in complete remission were sorted by flow cytometry and examined for co-expression of CD33 and CD11b. Fluorescence in situ hybridization (FISH)
The assay was used to examine 300 cells early in remission. As with control APL cells,
The majority of these cells exhibited a hybrid signal. This suggests that translocation between the PML gene and the RAR-α gene has occurred and that these signals are from neoplastic clones. However, when cells from the same patient were sorted again using the same parameters in late remission, only the normal pattern of the fluorescent signal was detected. Thus, it is suggested that these cells were derived from normal hematopoietic progenitor cells.

[0110] Western blot analysis: Protein extracts from bone marrow mononuclear cells were examined sequentially by Western blot analysis. This analysis showed that the precursor forms of caspase 2 and caspase 3 were up-regulated in vivo in response to arsenic trioxide treatment. Furthermore, this treatment induced the expression of a cleaved caspase 1 fragment, suggesting that the enzyme was activated. It is also suggested that the expression of cleaved caspase 3 is increased. The antibodies used in these experiments do not react with cleaved caspase-2.

6.3 Discussion In this study, with few exceptions, patients who participated in the study had multiple relapses and tolerated conventional chemotherapy, retinoid, or bone marrow transplantation. At the outset, patients enrolled in the study suffered from various leukemia-related complications, such as respiratory failure, disseminated varicella-zoster infection, cavernous aspergillosis, chronic renal failure, and graft-versus-host disease. Was. In addition, five of the twelve patients had to be placed in intensive care units for assisted breathing and assisted nursing, but these complications were not directly related to arsenic trioxide therapy. Was.

[0112] Virtually all patients diagnosed as having APL were in remission without premature death associated with retinoid therapy. Although arsenic trioxide induced significant leukocytosis in some patients, in fewer cases compared to all-trans-retinoic acid treatment. With the discontinuation of other cytotoxic drugs, leukocytosis disappeared as the patient remitted. Eight of the 11 patients tested had three initial relapses, but the RT-PCR assay for PML / RAR-α (a molecular marker of residual disease) turned negative. This change is a phenomenon not normally seen after all-trans-retinoic acid treatment alone. Ultimately, arsenic trioxide contains 0.06-0.20 mg
APL shows activity when administered at least 3-fold in the range of / kg.

Although all trans-retinoic acid induces “final” differentiation of APL cells, the cell differentiation effects of arsenic trioxide are not fully exhibited. Arsenic induces cell populations that simultaneously express unique surface antigens on both mature and immature cells (ie, CD11b and CD33, respectively). Early in induction, these induced cells maintain the t (15; 17) translocation that characterizes APL. Surprisingly, these cells persist in the bone marrow despite complete clinical remission. However, in late remission, these co-expressing cells, while still easily detectable, were not positive by in situ hybridization. Also, distinguishing the morphological appearance of leukemic cells during treatment is more difficult than during treatment with all-trans-retinoic acid. In fact, leukemic cells from many patients showed little morphological change for more than 10 days, after which the percentage of leukemic cells gradually decreased.

After “non-terminal” differentiation, arsenic trioxide appears to induce apoptosis. This is consistent with increased expression of cysteine protease (called caspase) and conversion of its inactive precursor to the active enzyme. The caspase pathway has only recently been characterized as a key pathway for programmed cell death. The family of caspases was first recognized by homology between C. elegans protein ced-3 and mammalian interleukin lβ converting enzyme (ICE), and now includes at least 10 different proteins that cleave a number of polypeptides. . In leukemic cell lines, several cytotoxic agents, such as all-trans-retinoic acid, can be used to trigger caspase activation. Since these enzymes induce extensive proteolysis, PML / RAR-α is considered to be a caspase substrate.

The final similarity shared by arsenic trioxide and all trans-retinoic acid is that
Clinical resistance is a sudden manifestation in some patients. Leukemia cells from two relapsed patients remained susceptible in vitro over a concentration range of 10 ⁻⁴ M to 10 ⁻⁷ M. Relative arsenic resistance based on reduced intracellular trafficking is
It has been described in connection with down-regulation of the membrane transporter encoded by the ars operon. Although resistance has not been well characterized in mammalian cells, alterations in membrane trafficking or efflux are probably important factors. In summary, arsenic trioxide elicits complete remission in APL patients who have relapsed after extensive prior treatment. This agent causes partial and incomplete differentiation of leukemic cells, followed by activation of caspases and induction of apoptosis.

[0116]

[Table 3]

All patients had previously received one or more administrations of all-trans-retinoic acid + anthracycline antibiotic + cytosine arabinoside. * Indicates patients found to have retinoid resistance (ie, no response upon reinduction or relapse despite retinoid retention). † indicates patients who died early. Other treatments: ^a Mitoxantrone / etoposide; ^b Allogeneic bone marrow transplantation; ^c Methotrexate / Vincristine / 6-mercaptopurine; ^d 9-cis-retinoic acid + M195 (anti-CD33 monoclonal antibody).

7. Examples: Clinical Use in Lymphoma Based on the first discovery of the antitumor effect of arsenic trioxide on the B-cell lymphocyte lineage in vitro, we One patient with moderate large cell lymphoma that relapsed after conventional treatment of the form was treated. Arsenic trioxide Treatment with arsenic trioxide significantly reduced the size of cancerous lymph nodes and spleen (> 50%) before starting treatment, despite the patient's disease progressing rapidly . This has also led to a significant improvement in the patient's quality of life.

8. Examples: Clinical use in non-hematopoietic cancers [0119] Arsenic trioxide was also used to treat colon cancer. In a preliminary study, one colon cancer patient treated once with arsenic trioxide had significantly reduced serum CEA (carcinoembryonic antigen) levels. 0.1-5 mg arsenic trioxide / kg body weight per day was administered daily for 5 days by intravenous infusion. CEA levels changed from 19,901 ng / ml to 15,266 ng / ml. That is, a 23% decrease was observed. As is well known, a decrease in serum CEA levels is associated with an anti-tumor response. Clinical data can also confirm that arsenic trioxide can be used to treat other non-hematopoietic cancers such as colon cancer.

9.Example: pharmacokinetic study As in APL patients and patients with other hematological disorders_TwoO_ThreePharmacokinetics (PK) and
Several studies on dose changes were performed to determine their biological effects.
For APL patients, flow cytometry for immunophenotyping, fluorescence in situ
Hybridization (FISH) and apoptosis-related protein caspase
Bone marrow mononuclear cells were continuously monitored by 1, 2, and 3 Western blot expressions. Cells co-expressing CD11b and CD33 and confirming the t (15; 17) translocation by FISH analysis gradually increased during treatment and persisted even in early remission. As_TwoO _Three Also demonstrated in vivo expression of caspase 2 and caspase 3 proenzymes, and
It induced the activation of bothase-1 and caspase-3. Perform PK analysis of blood and urine
When the elemental arsenic (As) content was examined by means of As, As was found in the plasma and red blood cell fractions of whole blood.
It was found to be distributed in both. From parallel elimination curves
It was suggested that these two compartments could move freely between the two compartments, and that the peak value decayed with a half-life of about 60 minutes. The average AUC on day 1 was about 400 ng · hr / ml. Approximately 20% of the dose was recovered in urine within the first 24 hours.

Next, using a daily intravenous schedule at a dose level of 0.1-0.15 mg / kg of body weight per day for a total of 25 days for a total of 25 days per course of treatment, APL A dose change study was initiated in patients with other diseases. To date, 10 patients have been recruited. The breakdown is CLL patients (2), AML patients (3), lymphoma patients (4)
) And CML patients (1). Five patients were initially excluded from the study due to rapid progress, and five patients underwent a planned 25-day course. At this dose range, the drug proved to be well tolerated. Side effects include:
Skin rash, dizziness during injection, fatigue, and increased EKG QTc were included. Judging from the results of this ongoing study, clinical use of As ₂ O ₃ induces partial differentiation and apoptosis in APL, but the therapeutic effects of this drug are not limited to this disease. I understand that there is no.

The scope of the present invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are
It is intended to be included within the scope of the appended claims. Various publications are cited herein, the disclosures of which are all incorporated herein by reference.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Pandolphy, Pier, Paolo United States 10022 New York, New York, East 60T Street 303 (72) Inventors Gabrillob, Janis, El. United States 10028 New York, New York, East 86 T H Street 25F Term (Reference) 4C086 CB04 DA31 HA07 HA28 MA01 MA02 MA04 MA05 MA66 NA14 ZB26 ZB27 4C206 AA01 AA02 DA05 JB06 KA01 MA01 MA02 MA03 MA04 MA05 MA86 NA14 ZB26 ZB27

Claims (18)

[Claims] 1. A method of treating acute myeloid leukemia in a human, comprising administering a therapeutically effective amount of arsenic trioxide to the human with acute myeloid leukemia. 2. A method for treating chronic myeloid leukemia in a human, comprising administering a therapeutically effective amount of arsenic trioxide to the human with chronic myeloid leukemia. 3. A method of treating solid cancer in a human comprising administering to the human a solid cancer, a therapeutically effective amount of arsenic trioxide. 4. The method of claim 3, wherein the solid cancer is a gastrointestinal tract, soft tissue, esophagus, liver, stomach, colon, lung, skin, brain, bone, breast or prostate cancer. 5. A method of treating leukemia in a human that is resistant to treatment with a retinoid, wherein the human in need thereof is administered a therapeutically effective amount of arsenic trioxide or melarsoprole. A method comprising: 6. A method of treating leukemia, lymphoma or solid tumor in a human, comprising administering to a human in need thereof a therapeutically effective amount of melarsoprol. 7. The method according to claim 1, wherein about 2.5 to 4.5 mg of arsenic trioxide is administered per day.
, 2, 3, 4 or 5. 8. The method of claim 1, 2, 3, 4 or 5, wherein about 0.15 mg of arsenic trioxide per kg of human weight is administered per day. 9. The method of claim 1, wherein the arsenic trioxide is administered by intravenous infusion.
Or the method according to 5. 10. The method of claim 1, 2 or 5, wherein the administration of arsenic trioxide is repeated daily until bone marrow remission is observed in said human. 11. The process of pausing treatment for 3 to 6 weeks and resuming daily administration of arsenic trioxide 5 to 7 times per week for a cumulative total of 25 days, 1 to 10 times 11. The method of claim 10, further comprising: 12. The method of claim 1, 2 or 5, wherein all trans retinoic acid is also administered to said human. 13. The method according to claim 3, wherein the administration of arsenic trioxide is repeated daily for 5 days. 14. The method of claim 13, wherein the method is repeated once a month. 15. The human of the present invention comprises about 0.5 to 5 mg of melarsoprol per kg of human body weight.
7. The method of claim 6, wherein the method is administered daily. 16. A method for producing a sterile pharmaceutical composition suitable for human administration, comprising arsenic trioxide, comprising: (a) solubilizing arsenic trioxide in an aqueous solution having a pH of 12 or more; Neutralizing the arsenic trioxide solution to about pH 8.5 with hydrochloric acid; (c) diluting the arsenic trioxide solution from step (b) in a pharmaceutical carrier that lowers and stabilizes the pH to about 7; Sterilizing the pharmaceutical composition. 17. A sterile pharmaceutical composition suitable for injection into humans, comprising arsenic trioxide and dextrose in a pharmaceutically acceptable carrier. 18. The pharmaceutical composition according to claim 17, comprising 1 mg / ml arsenic trioxide.